A large number of naturally-occurring organisms have been found to produce useful products, the large scale production of which is desirable for research and commercial purposes. Once such product has been identified efforts are being made to develop production methods leading to a high production of the product. One widely used method, which is based on recombinant DNA techniques, is to clone a gene encoding the product, inserting the gene into a suitable expression system permitting the expression of the product and culturing a suitable host cell comprising the expression system, either integrated in the chromosome or as an extrachromosomal entity, under conditions conducive for the expression of the product. However, a prerequisite for using such method is that the gene in question may be identified and cloned, and further that a suitable expression system and host cell for the production are available.
Another approach which may be used for the production of such products is to culture the cell which in nature produces the product or a derivative of such cell under suitable conditions. However, a frequently recognized drawback of such method is that the cell is not a suitable production organism, one reason being that the amount of product produced by such cell is too low to be commercially attractive.
Irrespective of which production method is used, it is normally desirable to be able to increase the production level of a given protein. Thus, efforts are being made to increase the production, e.g. by inserting the gene encoding the product under the control of a strong expression signal, or by increasing the number of copies of the gene in the production organism in question. This latter approach may be accomplished by inserting the gene into a multicopy plasmid which generally, however, tends to be unstable in the host cell in question, or by integrating multiple copies of the gene into the chromosome of the production organism, an approach which generally is considered more attractive because the stability of the construct tend to be higher allowing the gene to be stably maintained in the production organism.
EP 0 284 126 and EP 166 628 disclose methods for stably integrating one or more copies of a gene into the chromosome of a prokaryotic cell already harbouring at least one copy of the gene in question in its chromosome. According to EP 0 284 126, a host cell comprising said gene is transformed with a DNA construct comprising another copy of the gene, whereby, after a suitable selection procedure, a cell is obtained which in its chromosome comprises two copies of the gene separated by an endogenous chromosomal sequence which is vital to the host cell and thereby ensures stable maintenance of the integrated gene. This procedure may be repeated so as to produce cells harbouring multiple copies of the gene in its chromosome.
EP 166 628 relates to a process for amplifying a specific gene in the chromosome of a Bacillus strain thereby obtaining a cell harbouring a so-called "amplifiable unit" comprising the gene, the expression elements of the gene, and a gene encoding a selection marker inserted between two directly repeating sequences termed "duplicated sequences". The gene is introduced into the cell by a plasmid integration vector which is integrated in the Bacillus chromosome and which harbours a marker gene, the gene to be amplified, and one of the duplicated sequences, the other being present on the chromosome of the Bacillus cell.
Both of the above described methods require that the entire gene to be amplified is insertable into the vector to be used in the amplification method and thus, are applicable only when the gene to be amplified is isolated and available on a vector to be used in the method.